Co-culture of H295R Adrenocortical Carcinoma and BeWo Choriocarcinoma Cells to Study Feto-placental Interactions: Focus on Estrogen Biosynthesis
Andrée-Anne Hudon Thibeault, J. Thomas Sanderson, and Cathy Vaillancourt
Abstract
Estrogens are produced in large amounts during pregnancy, as a result of a tightly regulated cooperation between the maternal and fetal adrenal cortex, which produce androgen precursors, and the placental vil- lous trophoblast, which transforms these precursors into estrogens. These estrogens play an important role in proper placental function, in adaptation of the mother to pregnancy, as well as in adequate fetal develop- ment. Disruption of estrogen production is associated with poor pregnancy outcomes and fetal malforma- tion or altered fetal programming. Pregnant women may be exposed to endocrine disruptors from environmental sources or medications, and it is crucial to study the effects of such compounds on feto- placental steroidogenesis. The H295R/BeWo co-culture model offers the opportunity to study these interactions, by making it possible to evaluate the effects of chemical exposures on androgen and estrogen biosynthesis, as well as on various other aspects of feto-placental communication.
Key words Steroidogenesis, Feto-placental unit, Estrogen, Co-culture, Trophoblast, Fetal adrenocortical
1 Introduction
In the human placenta, the villous trophoblast produces large amount of estrogens, including pregnancy-specific estriol. This synthesis is dependent on precursors produced by the adrenal cor- tex of the mother and that of the developing fetus. We have previ- ously shown that the H295R/BeWo co-culture is a relevant model to study estrogen biosynthesis and fulfills an essential need as research tool considering the lack of suitable in vivo or ex vivo human models. In the co-culture model, H295R cells possess fetal adrenocortical characteristics, including the ability to synthetize
J. Thomas Sanderson and Cathy Vaillancourt share joint senior authorship and contributed equally to this work.
16-a-hydroxylated androgens [1]. BeWo cells are used as a villous trophoblast model since they possess high aromatase (CYP19) activity, which confer them the capacity of transforming androgens to estrogens in great amounts.
The H295R/BeWo co-culture model was first characterized for its capacity to reproduce the feto-placental estrogen biosyn- thetic profile with interactions between the two cell types occur- ring in real time. Moreover, culturing BeWo cells on porous inserts allows the cell to adopt a polarized phenotype with differential expression of proteins on the fetal membrane and maternal mem- brane [2-4], which opens up the possibility of studying feto- placental transport. The co-culture model also offers the possibility to study several other feto-placental interactions (stress hormones, IGF axis, drug metabolism, etc.) as summarized in Table 1.
| Placenta hormone/factor | Effect on fetal compartment | Reference |
|---|---|---|
| Placental growth hormone (pGH) or human placenta lactogen (hPL) | Stimulation of insulin-like growth factor (IGF) axis, which stimulates growth of the fetal adrenal cortex | [6] |
| Estradiol, progesterone, and prostaglandins | Regulation of the expression of the enzyme 11ß-hydroxysteroid dehydrogenase type 2 (HSD11B2) which oxidizes cortisol to cortisone, a major regulator of fetal organ maturation | [6-9] |
| Estradiol and ß-human chorionic gonadotropin (ß-hCG) | Regulation of DHEA sulfate synthesis, a precursor for estrogen production, in the H295R cells (REF) | [7,8] |
| Estrogens | Development and maturation of fetal adrenal gland (expression of steroidogenesis enzymes) Sexual differentiation Fetal growth | [9-19] |
| Corticotropin-releasing hormone (CRH) | Stimulation of cortisol and DHEA production | [20-22] |
| Epidermal growth factor (EGF) | Stimulation of fetal adrenal cell proliferation | [23] |
| Fetal adrenocortical hormone | Effects on placental compartment | Reference |
| Aldosterone | Stimulation of proliferation of trophoblast | [24] |
| Cortisol | Inhibition proliferation of trophoblast | [24] |
| Dehydroepiandrosterone (DHEA) | Regulation of implantation (mouse) | [25] |
2 Materials
2.1 Cell Culture
1. H295R medium: Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 without phenol red supplemented with 1.2 g/L sodium bicarbonate (NaHCO3), 2 mg/L pyridoxine·HCl, 2.5% Nu-serum, 1% insulin, transferrin, selenium (ITS) + premix.
2. BeWo medium: Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 without phenol red supplemented with 0.6 g/L sodium bicarbonate (NaHCO3) and 10% fetal bovine serum (FBS).
3. Co-culture medium: Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 without phenol red supplemented with 1.2 g/L sodium bicarbonate (NaHCO3), 2 mg/L pyridoxine·HCl, 2.5% Nu-serum, 1% insulin, transferrin, selenium (ITS) + premix, and 1% stripped FBS.
4. 24-well cell culture plate and polycarbonate transwell perme- able support with 0.4 M pores.
5. Phosphate-buffered saline (PBS).
6. TrypLE Express.
2.2 Real-Time Monitoring of Cell Proliferation
1. The xCELLigence Real-Time Cell Analyser Single Plate (RTCA-SP, see Note 1) (ACEA Biosciences).
2. Eplate and Eplate inserts (ACEA).
2.3 Aromatase Catalytic Activity
1. Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 without phenol red supplemented with 54 nM [16-3H(N)] androst-4-ene-3,17-dione.
2. 12-well plate.
3. Chloroform.
4. Dextran-coated charcoal (5% charcoal + 0.5% dextran (w/v)) (see Note 2).
5. Scintiverse BD scintillation cocktail.
6. MicroBeta TriLux liquid scintillation counter.
7. Liquid scintillation counting flexible microplates in 96-well and 24-well format.
8. Plate seal.
9. Uncompleted culture medium: Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 without phenol red.
10. Radioimmunoprecipitation assay buffer (RIPA): 150 mM NaCl, 50 mM Tris-HCI PH 7.1, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate.
11. Pierce BCA Protein Assay.
| 12. Powder weighing spatula (for cell scraping). 13. 70% ethanol. | |
|---|---|
| 2.4 Hormone Assay | Commercial enzyme-linked immunosorbent assays (ELISA) kits for ß-hCG, dehydroepiandrosterone, androstenedione, estrone, estradiol, estriol, and progesterone. |
| 2.5 Transepithelial Resistance | 1. Epithelial voltohmmeter (World Precision Instruments). 2. Bleach. |
| 3. 1.5 M KCl solution. | |
| 3 Methods | |
| 3.1 Cell Co-culture (See Note 3) | 1. Use 90% confluent 75 cm2 flasks of BeWo and H295R cells. Proceed one cell line at a time. 2. Discard the culture medium and rinse with 5 mL of PBS. 3. Trypsinize with 2 mL of TrypLE, until cells are detached. 4. Complete with 8 mL of regular culture medium for each cell type, respectively. |
| 5. Mix by pipetting up and down and determine cell concentration. 6. Seed H295R cells in a 24-well plate at 25,000 cells/well den- sity, 1 mL/well. | |
| 7. In another plate, seed BeWo cells in transwell inserts at 12,500 cells/well density, 0.2 mL/insert. Add 0.8 mL culture medium to the wells underneath the inserts. | |
| 8. Incubate at 37 ℃ for 24 h for cell adhesion. | |
| 9. Remove BeWo culture medium from the transwell inserts and wells and rinse cells twice with co-culture medium to remove FBS (see Note 4). | |
| 10. Remove H295R culture medium from the wells containing H295R cells. Rinsing is not necessary since H295R medium does not contain FBS. | |
| 11. Assemble the co-culture: 0.8 mL of co-culture medium in the wells and 0.2 mL in the transwell inserts (see Note 5). 12. Incubate at 37 ℃ for 24 h or longer according to the type of experiment (see Note 6). | |
| 3.2 Real-Time Monitoring of Cell Proliferation | 1. Set the xCELLigence software schedule in two steps: step 1, background measurement (default), and step 2, 10 min sweeps for at least 96 h. |
| 2. Add 50 µL of regular culture medium/well in an Eplate (96 well for SP instrument) and measure background (step 1). |
3. To the wells of the Eplate, add 100 µL of either BeWo (10,000 cells/well) or H295R (20,000 cells/well) cells.
4. Let the cells settle for 30 min at room temperature.
5. To the Eplate inserts, add 50 HL of BeWo (10,000 cells/insert) or H295R (20,000 cells/insert) cells. Add 130 µL of culture medium to the receiver plate.
6. Monitor cell proliferation in the Eplate (step 2) and incubate the receiver plate with the inserts containing cells for 24 h at 37 °C.
7. Pause step 2 and remove the Eplate from the instrument.
8. Rinse wells and inserts containing BeWo medium twice with co-culture medium, before adding the co-culture medium containing the test compound.
9. Assemble the co-culture by placing the inserts containing BeWo cells above the H295R cells in wells or vice versa.
10. Continue monitoring cell proliferation (resume step 2).
3.3 Aromatase Catalytic Activity (Tritiated Water-Release Assay)
1. After the treatment period (see Note 7), remove co-culture medium from the co-culture (see Note 8).
2. Place the inserts in a 12-well plate so that the bottom of the insert is in direct contact with the well.
3. Rinse inserts and wells twice with PBS.
4. Add 50 µL/insert or 250 µL/well of 54 nM [16-3H(N)] androst-4-ene-3,17-dione in uncompleted culture medium (Table 2).
5. Incubate 1.5 h at 37 °C.
| Insert Well | ||||
|---|---|---|---|---|
| Volume | Dilution factor | Volume | Dilution factor | |
| Working solution of [1ß-3H(N)] androst-4-ene-3,17-dione (54 nM) | 50 µL | 50/40 | 250 HL | 250/200 |
| Volume of supernatant + chloroform | 40 µL + 100 µL | 100/40 | 200 µL + 500 µL | 500/200 |
| Volume of supernatant + dextran-coated charcoal | 20 µL + 20 µL | 40/20 | 100 µL + 100 µL | 200/100 |
| Volume of supernatant + scintillation cocktail | 20 µL + 100 µL | 120/20 | 100 μL + 1000 μL 24 well | 1100/100 |
| Counting microplate | 96 well | |||
| Final dilution factor | 37.5 | 68.75 | ||
6. Take 40 L/insert or 200 µL/well of supernatant and place in a microtube containing 100 µL or 500 µL chloroform, respec- tively (Table 2).
7. Vortex and centrifuge at 12,000 x g for 5 min.
8. Take 20 µL/insert or 100 µL/well of supernatant and place in microtubes containing 20 µL or 100 µL dextran-coated char- coal, respectively (Table 2).
9. Vortex and incubate 5 min at room temperature and centri- fuge at 12,000 × g for 15 min.
10. Take 20 µL/insert or 100 µL/well of supernatant and place in a 96-well or 24-well liquid scintillation counting microplate, respectively (Table 2).
11. Add 10 µL of 54 nM [1-3H(N)]androst-4-ene-3,17-dione in uncompleted culture medium in a well of the liquid scintilla- tion counting microplate to determine the specific activity in the co-culture medium (Table 2).
12. Add 100 µL/insert or 1000 µL/well of liquid Scintiverse BD scintillation cocktail.
13. Count each well for 2 min in a MicroBeta TriLux liquid scintil- lation counter.
14. To assess protein content, rinse cells in the wells and inserts twice with cold PBS. Scrape cells in the wells and inserts (50 µL of RIPA/well) with powder weighing spatulas. Wash spatulas with 70% ethanol between samples. Vortex every 5 min during 30 min. Centrifuge 10 min at 21,000 x g. Use the supernatant for protein quantification following manufacturer’s instruction (Pierce BCA Protein Assay).
15. Results are either expressed as percentage of control or in pmoles of androstenedione converted/hour (Fig. 1). This expression can be normalized to mg protein content of the well or cell number.
3.4 Hormone Assay
1. Use the supernatant from the co-cultures and monocultures (see Note 9).
2. Keep the supernatants at -80 ℃ until use in the commercial kits, following manufacturer’s instructions (see Note 10).
3.5 Transepithelial Resistance
1. Rinse the electrodes with 70% ethanol in a 15 ml tube for 10 min.
2. Remove the electrodes from the ethanol and let dry for 15 s.
3. Place the electrodes in tubes containing pre-warmed (37 ℃) culture medium for at least 5 min.
4. Remove the BeWo culture medium and change to co-culture medium in the co-culture and BeWo monoculture, and place
i) Conversion of count per minute (CPM) to disintegration per minute (DPM)
Considering a quenching of 15%, DPM = CPM x 1.15
ii) Conversion of DPM to pmol
This conversion will depend on the specific activity of your [1]-3H(N)] androst-4-ene- 3,17-dione.
Example of conversion of [1B-3H(N)] androst-4-ene-3,17-dione at 26.3 Ci/mmol to DPM/mmol
26.3 Ci 3.7 x 1010 Disintegration per second (DPS) x
60 sec
1 mmol
58 386 DPM
mmol
Ci
x min ^109 pmol x
pmol
DPM
58 386DPM pmol
= pmol
iii) Expression considering the 3H position on androst-4-ene-3,17-dione
74.2% of tritiated-hydrogen (3H) is situated in ß position in the substrate [1]-3H(N)] androst-4-ene-3,17-dione, which will lead to tritiated water release when converted.
Tritiated-water
CH,
0
0
CH,
0
3H
PH
H
CH,
a
B
0
HO
[1]-3H(N)] androst-4-ene-3,17-dione
Estrone
pmol 0.742
= pmolcorrected
iv) Dilution factor during the extraction
The dilution factors are presented in table 1.
For inserts, pmolcorrected x 37.5
For wells, pmolcorrected x 68.75
v) Correction for time of incubation with substrate [13-3H(N)] androst-4-ene-3,17-dione
pmolcorrected
= pmolcorrected/hour
1.5 hour
vi) Correction for protein content of the well or number of cells
pmolcorrected/hour
= pmol/hour/cell number or pmol/hour/ mg protein
Cell number or protein content
Fig. 1 Conversion of CYP19 activity in count per minute (CPM) to pmol of androstenedione converted/hour
BeWo cells
H295R cells
the electrodes in the well and insert. The longest electrode has to touch the bottom of the well, passing through the side hole of the insert. The electrodes should stay immobile during the measurement (Fig. 2).
5. Measure 4 h after seeding the cells, directly after assembling the co-culture and every 24 h after assembly (see Note 11).
6. Place the electrode in co-culture medium between each mea- surement. At the end, place the electrodes in bleach for 3 min.
7. Rinse with water.
8. Between experiments, leave the electrodes in a KCl solution.
4 Notes
1. The instrument RTCA dual plate (DP) could also be used with the Eplate16. The well formats are identical, but with the DP instrument, there are only 16 wells/plate instead of 96 wells/ plate with the SP.
2. Prepare and mix the solution overnight on a magnetic stir plate and mix by inverting the bottle five times before using.
3. We recommend comparing the responses of the co-culture with those of BeWo and H295R cells in monoculture.
4. To rinse the inserts, prepare wells with 0.8 mL of co-culture medium. Remove the medium from each insert as well as under the insert, where a droplet often forms, and place it in the well containing co-culture medium. Add co-culture medium to the insert and repeat.
5. In order to have an even exposure of the cells to the test com- pound, it should be dissolved in culture medium before treat- ment instead of adding the compound directly to the culture medium in the well and insert.
6. We recommend a 24-h incubation of the co-culture, since a decrease in H295R cell proliferation is observed after 24 h under untreated circumstances [5].
7. To determine specificity of the tritiated water-release assay for aromatization, an irreversible inhibitor of the catalytic activity of aromatase, formestane (4-hydroxyandrostenedione), (1 µM) should be used. Positive controls (CYP19 inducers), such as forskolin (10 µM) or phorbol-12-myristate-13-acetate (1 µM), should also be included to determine the responsiveness of the cells.
8. The culture medium from the insert and well can be placed in microtubes at -80 ℃ for later hormone assay. Medium from the insert and well may be mixed together (total volume of 1 mL) or harvested separately, depending on the experimental requirements.
9. If too much cell debris is present, the culture medium may be centrifuged for 5 min at 10,000 x g.
10. Estrogen production by the untreated co-culture should be determined as a quality control. A synergistic production of estrogens and the presence of estriol should be observed in the co-culture compared to BeWo and H295R cells in monoculture.
11. Measurements will vary depending on the experiment. Values should always be compared within the same experiment. We consider that cells form a confluent monolayer when transepi- thelial resistance reaches a plateau.
Acknowledgments
This work was financially supported by the Réseau de recherche en santé environnementale as part of the Fonds de recherche du Québec (FRQ)-Santé (C.V., J.T.S.), the Natural Sciences and Engineering Research Council of Canada (NSERC) grants 313312-2012 (J.T.S.) and 262011-2009 (C.V.), the March of Dimes Foundation (C.V.), as well as studentship awards to A.A.H.T. from NSERC, FRQ-Nature et Technologies, FRQ- Santé, and Canadian Institutes of Health Research.
References
1. Gazdar AF, Oie HK, Shackleton CH, Chen TR, Triche TJ, Myers CE, Chrousos GP, Brennan MF, Stein CA, Larocca RV (1990) Establishment and characterization of a human adrenocortical carcinoma cell-line that
expresses multiple pathways of steroid- biosynthesis. Cancer Res 50:5488-5496
2. Poulsen MS, Rytting E, Mose T, Knudsen LE (2009) Modeling placental transport: correla- tion of in vitro BeWo cell permeability and
ex vivo human placental perfusion. Toxicol In Vitro 23:1380-1386
3. Prouillac C, Lecoeur S (2010) The role of the placenta in fetal exposure to xenobiotics: importance of membrane transporters and human models for transfer studies. Drug Metab Dispos 10:1623-1235
4. Audus KL (1999) Controlling drug delivery across the placenta. Eur J Pharm Sci 8:161-165
5. Hudon Thibeault AA, Deroy K, Vaillancourt C, Sanderson JT (2014) A unique co-culture model for fundamental and applied studies of human fetoplacental steroidogenesis and inter- ference by environmental chemicals. Environ Health Perspect 122:371-377
6. Myatt L, Sun K (2010) Role of fetal mem- branes in signaling of fetal maturation and par- turition. Int J Dev Biol 54:545-553
7. Gell JS, Oh J, Rainey WE, Carr BR (1998) Effect of estradiol on DHEAS production in the human adrenocortical cell line, H295R. J Soc Gynecol Invest 5:144-148
8. Rao CV, Zhou XL, Lei ZM (2004) Functional luteinizing hormone/chorioninc gonadotropin receptors in human adrenal cortical H295R cells. Biol Reprod 71:579-587
9. Kaludjerovic J, Ward WE (2012) The interplay between estrogen and fetal adrenal cortex. J Nutr Metab 2012:1-12
10. Mastorakos G, Ilias I (2003) Maternal and fetal hypothalamic-pituitary-adrenal axes during pregnancy and postpartum. Ann N Y Acad Sci 997:136-149
11. Tsatsaris V, Malassiné A, Fournier T, Handschuh K, Schaaps J-P, Foidart J-M, Evain- Brion D (2006) Placenta humain. Gynécol Obstétr 42:1-23
12. Albrecht ED, Aberdeen GW, Pepe GJ (2005) Estrogen elicits cortical zone-specific effects on development of the primate fetal adrenal gland. Endocrinology 146:1737-1744
13. Lash GE, Ansari T, Bischof P, Burton GJ, Chamley L, Crocker I, Dantzer V, Desoye G, Drewlo S, Fazleabas A, Jansson T, Keating S, Kliman HJ, Lang I, Mayhew T, Meiri H, Miller RK, Nelson DM, Pfarrer C, Roberts C, Sammar M, Sharma S, Shiverick K, Strunk D, Turner MA, Huppertz B (2009) IFPA meeting 2008 workshops report. Placenta 30:S4-S14
14. Jeschke U, Richter D-U, Möbius B-M, Briese V, Myolonas I, Friese K (2007) Stimulation of progesterone, estradiol and cortisol in tropho- blast tumor BeWo cells by glycodelin A N-glycans. Anticancer Res 27:2101-2108
15. Albrecht ED, Bonagura TW, Burleigh DW, Enders AC, Aberdeen GW, Pepe GJ (2006) Suppression of extravillous trophoblast invasion
of uterine spiral arteries by estrogen during early baboon pregnancy. Placenta 27:483-490
16. Gambino YP, Maymo JL, Perez Perez A, Calvo JC, Sanchez-Margalet V, Varone CL (2012) Elsevier Trophoblast Research Award lecture: molecular mechanisms underlying estrogen functions in trophoblastic cells - focus on leptin expression. Placenta 33:S63-S70
17. Olwenn MV, Shialis T, Lester JN, Scrimshaw MD, Boobis AR, Voulvoulis N (2008) Testicular dysgenesis syndrome and the estro- gen hypothesis: a quantitative meta-analysis. Environ Health Perspect 116:149-157
18. Toppari J, Virtanen HE, Main KM, Skakkebaek NE (2010) Cryptorchidism and hypospadias as a sign of testicular dysgenesis syndrome (TDS): Environmental connection. Birth Defects Res A Clin Mol Teratol 88:910-919
19. Dumitrescu A, Aberdeen GW, Pepe GJ, Albrecht ED (2014) Placental estrogen sup- presses cyclin D1 expression in the nonhuman primate fetal adrenal cortex. Endocrinology 155:4774-4784
20. Sirianni R, Rehman KS, Carr BR, Parker CR, Rainey WE (2005) Corticotropin-releasing hor- mone directly stimulates cortisol and the cortisol biosynthetic pathway in human fetal adrenal cells. J Clin Endocrinol Metabol 90:279-285
21. Sirianni R, Mayhew BA, Carr BR, Parker CR, Rainey WE (2005) Corticotropin-releasing hormone (CRH) and urocortin act through type 1 CRH receptors to stimulate dehydroepi- androsterone sulfate production in human fetal adrenal cells. J Clin Endocrinol Metabol 90:5393-5400
22. Smith R, Mesiano S, Chan E-C, Brown S, Jaffe RB (1998) Corticotropin-releasing hormone directly and preferentially stimulates dehydro- epiandrosterone sulfate secretion by human fetal adrenal cortical cells. J Clin Endocrinol Metabol 83:2916-2920
23. Riopel L, Branchaud CL, Goodyer CG, Zweig M, Lipowski L, Adkar V, Lefebvre Y (1989) Effect of placental factors on growth and func- tion of the human fetal adrenal in vitro. Biol Reprod 41:779-789
24. Gennari-Moser C, Khankin EV, Schuller S, Escher G, Frey BM, Portmann CB, Baumann MU, Lehmann AD, Surbek D, Karumanchi SA, Frey FJ, Mohaupt MG (2011) Regulation of placental growth by aldosterone and corti- sol. Endocrinology 152:263-271
25. Frolova AI, O’Neill K, Moley KH (2011) Dehydroepiandrosterone inhibits glucose flux through the pentose phosphate pathway in human and mouse endometrial stromal cells, preventing decidualization and implantation. Mol Endocrinol 25:1444-1455